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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Division of Hematology, the Institute of
Clinical Medicine, and the College of Medical Technology, University of
Tsukuba, Tsukuba, Ibaraki, Japan.
We studied the role of adenosine (Ado), which is generated from
adenine nucleotides via the activity of ecto-5'-nucleotidase (ecto-5'-NT), in the inhibition of platelet aggregation by
endothelial cells (ECs). The enzymatic activity of nucleotidases on
human umbilical vein endothelial cells (HUVECs) was examined with
regard to (1) the inhibition of adenosine diphosphate (ADP)-induced
platelet aggregation and (2) the liberation of inorganic phosphate from adenine nucleotides. Adenosine 5'-monophosphate (AMP) preincubated with HUVECs significantly inhibited ADP-induced platelet aggregation. This was completely blocked by the treatment of HUVECs with a specific
inhibitor of ecto-5'-NT, 5'-[ It has been well established that quiescent
endothelial cells (ECs) exert anticoagulant effects both through their
expression of thrombomodulin1 and heparan sulfate
proteoglycans2 and by the release of tissue factor pathway
inhibitor.3 ECs also inhibit platelet aggregation through
the production of nitric oxide (NO)4 and prostacyclin
(PGI2)5 and through degradation of adenosine
diphosphate (ADP) by adenosine triphosphate (ATP) diphosphohydrolase
(ATPDase), which is expressed on the luminal surface of
ECs.6-8 However, ATPDase can inhibit platelet aggregation and recruitment in the absence of NO and
PGI2.6-8 ADP released from activated platelets
induces platelet recruitment followed by further platelet aggregation
via binding to platelet P2X, P2T, and
P2Y receptors.9,10 ATPDase (molecular mass,
70-100 kd) is a glycoprotein belonging to the E-type ATPase
family.11 This enzyme hydrolyzes ATP and ADP to ADP and
adenosine 5'-monophosphate (AMP), respectively. Enzyme activity is
dependent on calcium (Ca2+) and magnesium
(Mg2+), and is inhibited by chelating agents, azides, and
ATP analogues, although it is not affected by inhibitors of P-, F- and
V-type ATPases.6,8,11,12 Degradation of extracellular ADP
by ATPDase has been recognized as important for the inhibition of
platelet aggregation by ECs, because of the critical role ADP plays as an agonist for platelet aggregation.6-8,13
Ecto-5'-nucleotidase (ecto-5'-NT) (CD73) also participates in adenine
nucleotide metabolism on the surface of ECs.14,15 AMP, generated from ADP by the action of ATPDase, is
subsequently hydrolyzed to adenosine (Ado) by ecto-5'-NT. Widely
distributed in bacteria, plant cells, and vertebrate tissues, 5'-NT is
classified into 4 groups according to cellular location and biochemical
properties: a membrane-anchored ecto-5'-NT, a soluble form
derived from ecto-5'-NT, and 2 cytoplasmic forms. Ecto-5'-NT, anchored
to the plasma membranes via glycosyl-phosphatidylinositol (GPI)
moiety,16 is distributed in a variety of cells including
hepatocytes, fibroblasts, endothelial cells, lymphocytes, and glial
cells.17 The catalytic activity of 5'-NT controls
intracellular and extracellular levels of AMP and Ado, thereby allowing
Ado to be metabolized for the synthesis of adenine nucleotides in the
purine salvage pathway.18 In addition to its enzymatic
activity, ecto-5'-NT is involved in cell-cell and cell-matrix
interactions and transmembrane signaling.19,20
The disulfide-linked homodimer form of ecto-5'-NT is essential for its
enzymatic activity. Although ecto-5'-NT hydrolyzes a variety of
nucleoside 5'-monophosphates, it has greatest affinity for AMP,
with Km values in the micromolar range. Enzymatic activity is not
dependent on added divalent cations but is inhibited by metal ion
chelating agents because of the presence of several potential zinc
(Zn2+) binding sites that are important for its
activity.21 Adenosine 5'-[ Ado inhibits a variety of cellular functions including platelet
aggregation,23 expression of tissue factor or adhesion
molecules and cytokine release by activated ECs,24,25
neutrophil adherence and injury to ECs,26 and release of
superoxide from neutrophils.27 However, Ado also enhances
NO production by ECs.28 These effects are mediated through
the binding of Ado to the A1, A2, and
A3 receptors that are expressed on cells in a variety of
tissues. The inhibition of platelet aggregation by Ado is thought to be mediated by the stimulation of adenylate cyclase through
A2a receptors expressed on platelets.23
Although the inhibitory aggregation effect of Ado on platelet is
evident in vitro,23 its effect in vivo has remained
controversial because of its rapid transport into
cells29,30 and its rapid degradation by Ado deaminase (ADA).31 However, studies showing that both platelet
aggregation ex vivo and thrombosis formation in vivo were inhibited by
the administration of Ado analogues to humans and dogs32
and that an Ado receptor antagonist caused thrombosis in
dogs33 suggest a critical involvement of Ado in the
inhibition of platelet aggregation in vivo.
In the present study, we examined ecto-5'-NT activity on the surface of
human umbilical vein endothelial cells (HUVECs) and confirmed the
cooperative function of ecto-5'-NT with ATPDase in the regulation of
platelet aggregation.
Materials
Cell culture of HUVECs
Inhibition of platelet aggregation by ADP preincubated on HUVECs Nucleotidase activity. HUVECs were grown to confluence in a gelatin-coated 96-well plate. After removal of the culture medium, the HUVEC-containing wells and the cell-free (blank) wells were washed 3 times with 50 mmol/L phosphate-free buffer containing Tris HCl (tris[hydroxymethyl] aminomethane hydrochloride) buffer (pH 8.0) and 150 mmol/L sodium chloride (NaCl), 5 mmol/L calcium dichloride (CaCl2), 5 mmol/L magnesium dichloride (MgCl2), and 0.1 mg/mL BSA (buffer A). The cells were then incubated with 200 µL buffer A containing 1-75 µmol/L ADP at 37°C for 15 minutes. The amounts of inorganic phosphate (Pi) liberated into the supernatants were measured by the malachite green colorimetric assay as described by Baykov et al.35 The agonistic activity of the incubation buffer on platelets following incubation with HUVECs or blank wells was also examined as described below. In some experiments, HUVECs were treated with 0.1-10 mmol/L levamisole, an inhibitor of alkaline phosphatase,36,37 or 1-100 µmol/L APCP before incubation with ADP. Inhibition of platelet aggregation. Blood from healthy volunteers was drawn into a 0.1 volume of sodium citrate (3.8%). Platelet-rich plasma (PRP) was obtained by differential centrifugation of the blood. PRP containing 6 × 107 platelets per 200 µL was preincubated at 37°C in an aggregometer cuvette. Platelet activation was then started by the addition of 22 µL of an ADP-containing (1-75 µmol/L) buffer preincubated on the HUVECs. Platelet aggregation was monitored by the increase of light transmission using an aggregometer (Hema Tracer I, Niko Bio Science, Tokyo, Japan). The percentage of aggregation was defined as the maximal change of light transmission within 10 minutes. Inhibition of ADP-induced platelet aggregation by HUVECs exposed to AMP Nucleotidase activity. HUVECs were grown to confluence in a gelatin-coated 96-well plate. After removal of the medium, the HUVEC-containing wells or the blank wells were washed twice with the wash buffer (buffer B) containing 30 mmol/L Tris HCl (pH 7.4) with 0.25 mmol/L ethylenediamine tetraacetic acid (EDTA), 0.125 mmol/L ethyleneglycotetraacetic acid (EGTA), 130 mmol/L sodium chloride (NaCl), and 5.5 mmol/L glucose. The wells were then washed with the incubation buffer (buffer C) containing 50 mmol/L Tris HCl (pH 7.4) with 130 mmol/L NaCl, 5 mmol/L MgCl2, and 5.5 mmol/L glucose. Buffer C, containing 1-100 µmol/L AMP, was then added to the wells and incubated at 37°C for 15 minutes. The amounts of Pi liberated into the supernatants were measured as described above. The inhibitory effect of the preincubated buffer on HUVECs or blank wells on ADP-induced platelet aggregation was also examined as described below. In some experiments, HUVECs were treated with one of the following inhibitors prior to incubation with AMP: 0.1-10 mmol/L L-NAME, an inhibitor of NO synthase; 0.01-1 mmol/L aspirin, an inhibitor of cyclo-oxygenase5,38; 1-100 µmol/L DIP, an inhibitor of adenosine transporter39; 0.1-10 mmol/L levamisole; or 100 µmol/L APCP. Inhibition of ADP-induced platelet aggregation by HUVECs exposed to AMP. We added 22 µL of AMP-containing buffer (1-100 µmol/L) preincubated on HUVECs or blank wells to 200 µL PRP immediately before the activation of PRP by ADP (final concentrations, 2-3 µmol/L); then platelet aggregation was monitored as described above. In some experiments, 0.5-10 µmol/L CSC, a selective antagonist for the A2a receptor,40 was added to PRP with the incubation buffer from HUVECs or blank wells immediately before the activation of platelets by ADP. Production of NO and PGI2 by HUVECs The concentration of NO in the incubation buffer was measured as total nitrate/nitrite concentration using a nitrate/nitrite colorimetric assay kit. In some experiments, HUVECs were pretreated with 0.01-10 mmol/L L-NAME. The production of PGI2 during the incubation period was evaluated by measuring its stable breakdown products, 6-keto-prostaglandin F1 and
2,3-dinor-6-keto-prostaglandin F1 in the incubation
buffer using a urinary prostacyclin enzyme immunoassay kit. In some
experiments, HUVECs were pretreated with 0.001-1 mmol/L aspirin.
Statistical analyses The Student unpaired t test was used for all statistical analyses.
Inhibition of platelet aggregation by ADP preincubated on HUVECs ADP incubated on the blank wells did not release Pi and induced aggregation of platelets (Figure 1). However, Pi was liberated from ADP on HUVECs in a dose-dependent manner relative to the concentration of ADP added (Figure 1B). Platelet aggregation induced by the ADP-containing buffer preincubated on HUVECs was inhibited compared with aggregation induced by the buffer preincubated on blank wells (Figure 1).
Inhibition of ADP-induced platelet aggregation by AMP preincubated on HUVECs AMP was not hydrolyzed on blank wells (Figure 2B). Pi was liberated by the incubation of AMP on HUVECs in a dose-dependent manner relative to the concentration of AMP added (Figure 2B). The AMP-containing buffer preincubated on HUVECs inhibited ADP-induced platelet aggregation compared with that preincubated on blank wells (Figure 2).
Involvement of NO and PGI2 in the inhibition of ADP-induced platelet aggregation by AMP preincubated on HUVECs The 30 µmol/L AMP buffer preincubated on HUVECs did not contain detectable amounts of nitric oxide. Pretreatment with more than 10 µmol/L L-NAME, which completely inhibited NO production in the growth medium of HUVECs, did not affect the inhibition of ADP-induced platelet aggregation by the AMP buffer preincubated on HUVECs (Figure 3A). This buffer contained 0.76 ± 0.09 pg/105 cells per hour (the mean ± SD) of PGI2, which is at the lowest limit of detection of the assay used. Pretreatment with more than 10 µmol/L aspirin completely blocked PGI2 production in the growth medium of HUVECs. Under these conditions, inhibition of ADP-induced platelet aggregation by AMP buffer preincubated on HUVECs was not affected (Figure 3B).
Involvement of Ado receptor in the inhibition of ADP-induced platelet aggregation by AMP preincubated on HUVECs In the presence of CSC, inhibition of ADP-induced platelet aggregation by purified Ado was abrogated in a dose-dependent manner (data not shown). The inhibition of ADP-induced platelet aggregation by 30 µmol/L AMP buffer preincubated on HUVECs was blocked by more than 1 µmol/L CSC (Figure 3C).Involvement of ecto-5'-NT in the inhibition of ADP-induced platelet aggregation by AMP preincubated on HUVECs Levamisole (10 mmol/L) did not affect AMP degradation on HUVECs (data not shown). APCP inhibited AMPase activity on HUVECs in a dose-dependent manner, as measured by the mean ± SD: Pi release without APCP, 21.0 ± 1.9 µmol/L; Pi release with 1 µmol/L APCP, 11.6 ± 2.2 µmol/L; Pi release with 10 µmol/L APCP, 5.6 ± 1.3 µmol/L. Pi was not liberated with 100 µmol/L APCP (Figure 4B). Inhibition of platelet aggregation by the AMP buffer preincubated on HUVECs was also attenuated by APCP in a dose-dependent manner (data not shown) and was completely blocked by 100 µmol/L APCP (Figure 4A).
Involvement of ecto-5'-NT in the inhibition of platelet aggregation by ADP preincubated on HUVECs Pretreatment of HUVECs with APCP partially inhibited Pi liberation from ADP preincubated on HUVECs (Figure 5B). Platelet aggregation induced by the ADP buffer preincubated on HUVECs was restored by the pretreatment of HUVECs with APCP (Figure 5A).
The ability of HUVECs to inhibit platelet aggregation by ADP degradation was confirmed6 (Figure 1). The ADP- or AMP-treated buffers preincubated on HUVECs could contain not only adenine nucleotides but also NO and PGI2 produced in the incubation periods. Because both NO and PGI2 are major substances released from ECs that inhibit platelet aggregation, it was possible that NO and/or PGI2 were involved in the mechanisms that inhibited platelet aggregation under our experimental conditions. HUVECs released NO in the growth medium at 0.14 ± 0.02 (mean ± SD) nmol/105 cells per hour, at the lowest limit of detection of the assay. However, we removed the medium followed by 3 washes of the wells prior to incubation with ADP- or AMP-containing buffer for 15 minutes. These incubation buffers did not contain any detectable amounts of nitric oxide. Pretreatment of HUVECs with more than 10 µmol/L L-NAME, which was enough to block NO production in the growth medium, did not change the inhibitory effect on platelet aggregation of ADP buffer preincubated on HUVECs (data not shown). This suggested that NO produced in the buffer during the incubation phase did not affect platelet aggregation under our experimental conditions. Basal levels of PGI2 in the growth medium of HUVECs were 1.34 ± 0.25 pg/105 cells per hour, and the incubation buffer contained 0.51 ± 0.07 pg/105 cells per hour, the limit of detection of the assay. Aspirin completely inhibited PGI2 production during the incubation of buffer at 0.01-1 mmol/L aspirin (data not shown). Because pretreatment of HUVECs with 1 mmol/L aspirin did not influence the inhibition of platelet aggregation by the ADP buffer preincubated on HUVECs (data not shown), it is likely that PGI2 in the incubation buffer did not affect the inhibitory effect on aggregation in our experiments. On the surface of HUVECs, ATPDase, ecto-5'-NT, and ALP can hydrolyze adenine nucleotides.11,14,37,41 However, pretreatment of HUVECs with 0.1-10 mmol/L levamisole, a specific inhibitor of ALP,36,37 did not affect Pi liberation from ADP in our experiments. This may be due to our experimental condition being at a lower pH than the optimal pH for ALP. Therefore, the role of HUVECs in the inhibition of platelet aggregation shown in Figure 1 could be explained as ADP degradation by nucleotidases on HUVECs. Undoubtedly, hydrolysis of ADP by ATPDase plays a pivotal role in the inhibition of platelet aggregation (Figure 1). However, we hypothesized that ecto-5'-NT was involved in this cooperative function with ATPDase because AMP generated by ATPDase is subsequently hydrolyzed to Ado by the action of ecto-5'-NT. In a preliminary experiment, we found that purified Ado, but not AMP, inhibited ADP-induced platelet aggregation in a dose-dependent manner in vitro and that this effect was completely blocked by CSC, a selective antagonist for the A2a receptor (data not shown). Previous studies showed that Ado inhibits platelet aggregation by binding to the A2a receptor on the platelets, thereby activating adenylate cyclase and elevating the cAMP level.23 We first examined enzymatic activity of ecto-5'-NT on HUVECs. Although AMP was not hydrolyzed on blank wells, Pi was liberated from AMP in a dose-dependent manner relative to the concentration of AMP added to HUVECs (Figure 2B). This AMP buffer preincubated on HUVECs inhibited ADP-induced platelet aggregation in proportion to the amounts of Pi liberated (Figure 2B). The incubation buffer could contain the remaining AMP, Ado generated, and several substances, including NO and PGI2, which were produced in the incubation phase. Our system did not contain strong agonists for either PGI2 or NO production. Any quantities that were produced were at baseline levels (Figure 3). The ineffectiveness of L-NAME on the function of AMP buffer preincubated on HUVECs to inhibit ADP-induced aggregation (Figure 3A) indicated that NO was not a major contributor under our experimental conditions. This might be due to the conditions of HUVECs in the confluent layer because previous studies revealed that NO synthase expression and NO release declined in the confluent phase.42 As for PGI2, AMP buffer preincubated on HUVECs contained little PGI2, and pretreatment of HUVECs with aspirin did not affect the inhibitory effects of the incubation buffer (Figure 3B). This suggests that PGI2 produced during the incubation phase did not influence ADP-induced platelet aggregation in our experiments. The IC50 value of PGI2 for ADP-induced platelet aggregation is about 100-fold higher than the concentration of the preincubated buffer.43 The production of PGI2 by ECs may decrease in the subculture and confluent states in our experiments, as previously shown in several studies.44,45 Because we hypothesized that the hydrolysis of AMP by ecto-5'-NT was the main mechanism by which AMP buffer preincubated on HUVECs inhibited ADP-induced platelet aggregation (Figure 2), we expected that the Ado receptor antagonists would block this effect. As expected, CSC, a selective A2a receptor antagonist,40 abrogated this effect in a dose-dependent manner (Figure 3C). Therefore, it is likely that the inhibitory effect of AMP buffer preincubated on HUVECs is derived via an interaction of Ado with the Ado receptor on platelets. The remaining question was whether some portions of Ado produced on the luminal surface may be taken up by HUVECs or deaminated by ADA before exerting any biological effects. Pretreatment of HUVECs with 1-100 µmol/L DIP, an inhibitor of Ado transport,39 did not affect AMP degradation or the inhibitory effect of the buffer on platelet aggregation (data not shown). This indicated that transport of Ado across the membranes had a limited effect on the extracellular levels of Ado, at least in our experimental conditions. Taken together with the report by Aalto et al,46 which showed that the effect of deamination of Ado by ADA on cultured HUVECs is limited, it is likely that in our experiments, the extracellular levels of Ado are regulated mainly by the generation of Ado from adenine nucleotides on HUVECs. Because treatment with levamisole, an inhibitor of ALP, did not interfere with the AMPase activity on HUVECs, we estimated that ecto-5'-NT was mainly involved in the hydrolysis of AMP. APCP, an ADP analogue modified on the phosphate chain by substituting a bridging oxygen with a methylene, is a specific inhibitor of 5'-NT (Ki = 6 nmol/L).14,22 It was demonstrated that APCP is neither affected by the hydrolysis of ADP by ATPDase nor is it hydrolyzed by ATPDase.47 APCP displayed almost no affinity for the P2X receptor,48 which was also supported by our findings that ADP-induced platelet aggregation was not influenced by the addition of APCP (data not shown). As shown in Figure 5, pretreatment of HUVECs with APCP resulted in a partial decrease in Pi liberation from HUVECs that were treated with ADP. In addition, platelet aggregation was partially restored at comparatively low concentrations of ADP. This might be explained by the fact that while a low concentration of ADP could be completely hydrolyzed by ATPDase on HUVECs, excessive ADP, which was undigested by ATPDase, inhibited the activity of ecto-5'-NT. Therefore, it seems reasonable to suppose that ecto-5'-NT is involved in the HUVEC function of inhibiting platelet aggregation in cooperation with ATPDase, especially at comparatively low concentrations of ADP. Inhibition of remnant ADP, which is not hydrolyzed by ATPDase, by Ado generated by the activity of ecto-5'-NT should lead to an increase in the threshold for platelet aggregation. In conclusion, we identified a contribution of ecto-5'-NT to the inhibitory effect of HUVECs on platelet agregation in cooperation with ATPDase at a comparatively low concentration of ADP. Ecto-5'-NT seems to be effective for prevention of thrombosis formation by increasing the threshold for platelet aggregation, although its role in vivo remains to be clarified. Results from these studies verify the concept that platelets in motion and in close proximity to endothelial cells do not respond to standard platelet agonists. This is probably due to the combined action of ATPDase and ecto-5'-NT described in our studies.
We thank Dr T. Kubo and all the doctors in the Division of Gynecology and Obstetrics, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan, for their generous provision of human umbilical cord.
Submitted September 8, 1999; accepted April 28, 2000.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Haruhiko Ninomiya, College of Medical Technology, University of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki 305-8577, Japan; e-mail: hninomiya{at}itan.tsukuba.ac.jp.
1.
Esmon CT.
The regulation of natural anticoagulant pathways.
Science.
1987;235:1348-1352 2. Marcum JA, Rosenberg RD. Anticoagulantly active heparin-like molecules from vascular tissue. Biochemistry. 1984;23:1730-1737[Medline] [Order article via Infotrieve]. 3. Broze GJ Jr. The role of tissue factor pathway inhibitor in a revised coagulation cascade. Semin Hematol. 1992;29:159-169[Medline] [Order article via Infotrieve].
4.
Ignarro LJ.
Nitric oxide: a novel signal transduction mechanism for transcellular communication.
Hypertension.
1990;16:477-483 5. Moncada S. Biological importance of prostacyclin. Br J Pharmacol. 1982;76:3-31[Medline] [Order article via Infotrieve]. 6. Marcus AJ, Safier LB, Hajjar KA, et al. Inhibition of platelet function by an aspirin-insensitive endothelial cell ADPase: thromboregulation by endothelial cells. J Clin Invest. 1991;88:1690-1696.
7.
Kaczmarek E, Koziak K, Sevigny J, et al.
Identification and characterization of CD39/vascular ATP diphosphohydrolase.
J Biol Chem.
1996;271:33116-33122 8. Marcus AJ, Broekman MJ, Drosopoulos JHF, et al. The endothelial cell ecto-ADPase responsible for inhibition of platelet function is CD39. J Clin Invest. 1997;99:1351-1360[Medline] [Order article via Infotrieve].
9.
Daniel JL, Dangelmaier C, Jin J, Ashby B, Smith JB, Kunapuli SP.
Molecular basis for ADP- induced platelet activation. Evidence for three distinct ADP receptors on human platelets.
J Biol Chem.
1998;273:2024-2029
10.
Hechler B, Léon C, Vial C, et al.
The P2Y receptor is necessary for adenosine 5'-diphosphate-induced platelet aggregation.
Blood.
1998;92:152-159 11. Plesner L. Ecto-ATPases: identities and functions. Int Rev Cytol. 1995;158:141-214[Medline] [Order article via Infotrieve]. 12. Komoszynski M, Wojtczak A. Apyrases (ATP diphosphohydrolases, EC 3.6.1.5): function and relationship to ATPases. Biochim Biophys Acta. 1996;1310:233-241[Medline] [Order article via Infotrieve]. 13. Gayle RB III, Maliszewski CR, Gimpel SD, et al. Inhibition of platelet function by recombinant soluble ecto-ADPase/CD39. J Clin Invest. 1998;101:1851-1859[Medline] [Order article via Infotrieve]. 14. Zimmermann H. 5'-nucleotidase: molecular structure and functional aspects. Biochem J. 1992;285:345-365. 15. Zimmermann H. Biochemistry, localization and functional roles of ecto-nucleotidases in the nervous system. Prog Neurobiol. 1996;49:589-618[Medline] [Order article via Infotrieve]. 16. Misumi Y, Ogata S, Ohkubo K, Hirose S, Ikehara Y. Primary structure of human placental 5'-nucleotidase and identification of the glycolipid anchor in the mature form. Eur J Biochem. 1990;191:563-569[Medline] [Order article via Infotrieve]. 17. Resta R, Hooker SW, Hansen KR, et al. Murine ecto-5'-nucleotidase (CD73): cDNA cloning and tissue distribution. Gene. 1973;133:171-177. 18. Thompson LF. Ecto-5'-nucleotidase can provide the total purine requirements of mitogen-stimulated human T cells and rapidly dividing human B lymphoblastoid cells. J Immunol. 1985;134:3794-3797[Abstract]. 19. Stochaj U, Mannherz HG. Chicken gizzard 5'-nucleotidase functions as a binding protein for the laminin/nidogen complex. Eur J Cell Biol. 1992;59:364-372[Medline] [Order article via Infotrieve].
20.
Airas L, Hellman J, Salmi M, et al.
CD73 is involved in lymphocyte binding to the endothelium: characterization of lymphocyte-vascular adhesion protein-2 identifies it as CD73.
J Exp Med.
1995;182:1603-1608 21. Knofel T, Strater N. X-ray structure of the Escherichia coli periplasmic 5'-nucleotidase containing a dimetal catalytic site. Nat Struct Biol. 1999;6:448-453[Medline] [Order article via Infotrieve]. 22. Naito Y, Lowenstein JM. 5'-nucleotidase from rat heart membranes: inhibition by adenine nucleotides and related compounds. Biochem J. 1985;226:645-651[Medline] [Order article via Infotrieve]. 23. Cristalli G, Volpini R, Vittori S, et al. 2-alkynyl derivatives of adenosine-5'-N-ethyluronamide: selective A2 adenosine receptor agonists with potent inhibitory activity on platelet aggregation. J Med Chem. 1994;37:1720-1726[Medline] [Order article via Infotrieve]. 24. Deguchi H, Takeya H, Urano H, Gabazza EC, Zhou H, Suzuki K. Adenosine regulates tissue factor expression on endothelial cells. Thromb Res. 1998;91:57-64[Medline] [Order article via Infotrieve].
25.
Bouma MG, van den Wildenberg FAJM, Buurman WA.
Adenosine inhibits cytokine release and expression of adhesion molecules by activated human endothelial cells.
Am J Physiol.
1996;270:C522-529 26. Cronstein BN, Levin RI, Belanoff J, Weissmann G, Hirschhorn R. Adenosine: an endogenous inhibitor of neutrophil-mediated injury to endothelial cells. J Clin Invest. 1996;78:760-770. 27. Roberts PA, Newby AC, Hallett MB, Campbell AK. Inhibition by adenosine of reactive oxygen metabolite production by human polymorphonuclear leucocytes. Biochem J. 1985;227:669-674[Medline] [Order article via Infotrieve].
28.
Li J-M, Fenton RA, Cutler BS, Dobson JG Jr.
Adenosine enhances nitric oxide production by vascular endothelial cells.
Am J Physiol.
1995;269:C519-523 29. Pearson JD, Carleton JS, Hutchings A, Gordon JL. Uptake and metabolism of adenosine by pig aortic endothelial and smooth-muscle cells in culture. Biochem J. 1978;170:265-271[Medline] [Order article via Infotrieve].
30.
Dieterle Y, Ody C, Ehrensberger A, Stalder H, Junod AF.
Metabolism and uptake of adenosine triphosphate and adenosine by porcine aortic and pulmonary endothelial cells and fibroblasts in culture.
Circ Res.
1978;42:869-876
31.
Moser GH, Schrader J, Deussen A.
Turnover of adenosine in plasma of human and dog blood.
Am J Physiol.
1989;256:C799-C806 32. Bullough DA, Zhang C, Montag A, Mullane KM, Young MA. Adenosine-mediated inhibition of platelet aggregation by acadesine: a novel antithrombotic mechanism in vitro and in vivo. J Clin Invest. 1994;94:1524-1532.
33.
Kitakaze M, Hori M, Sato H, et al.
Endogenous adenosine inhibits platelet aggregation during myocardial ischemia in dogs.
Circ Res.
1991;69:1402-1408 34. Jaffe EA, Nachman RL, Becker CG, Minick CR. Culture of human endothelial cells derived from umbilical veins. J Clin Invest. 1973;52:2745-2756. 35. Baykov AA, Evtushenko OA, Avaeva SM. A malachite green procedure for orthophosphate determination and its use in alkaline phosphatase-based enzyme immunoassay. Anal Biochem. 1988;171:266-270[Medline] [Order article via Infotrieve]. 36. Fallon MD, Whyte MP, Teitelbaum SL. Stereospecific inhibition of alkaline phosphatese by L-Tetramisole prevents in vitro cartilage calcification. Lab Invest. 1980;43:489-494[Medline] [Order article via Infotrieve]. 37. Harris H. The human alkaline phosphatases: what we know and what we don't know. Clin Chim Acta. 1989;186:133-150.
38.
de Witt DL, Smith WL.
Primary structure of prostaglandin G/H synthase from sheep vesicular gland determined from the complementary DNA sequence.
Proc Natl Acad Sci U S A.
1988;85:1412-1416 39. Fitzgerald GA. Drug therapy: dipyridamole. N Engl J Med. 1987;316:1247-1257[Medline] [Order article via Infotrieve]. 40. Jacobson KA, Nikodijevic O, Padgett WL, Gallo-Rodriguez C, Maillard M, Daly JW. 8-(3-chlorostyryl) caffeine (CSC) is a selective A2-adenosine antagonist in vitro and in vivo. FEBS Lett. 1993;323:141-144[Medline] [Order article via Infotrieve]. 41. Gallo RL, Dorschner RA, Takashima S, Klagsbrun M, Eriksson E, Bernfield M. Endothelial cell surface alkaline phosphatase activity is induced by IL-6 released during wound repair. J Invest Dermatol. 1997;109:597-603[Medline] [Order article via Infotrieve].
42.
Arnal J-F, Yamin J, Dockery S, Harrison DG.
Regulation of endothelial nitric oxide synthase mRNA, protein, and activity during cell growth.
Am J Physiol.
1994;267:C1381-C1388 43. Radomski MW, Palmer RMJ, Moncada S. Comparative pharmacology of endothelium-derived relaxing factor, nitric oxide and prostacyclin in platelets. Br J Pharmacol. 1987;92:181-187[Medline] [Order article via Infotrieve]. 44. Ager A, Gordon JL, Moncada S, Pearson JD, Salmon JA, Trevethic MA. Effects of isolation and culture on prostaglandin synthesis by porcine aortic endothelial and smooth muscle cells. J Cell Physiol. 1982;110:9-16[Medline] [Order article via Infotrieve].
45.
De Caterina R, Dorso CR, Tack-Gordman K, Weksler BB.
Nitrates and endothelial prostacyclin production: studies in vitro.
Circulation.
1985;71:176-182
46.
Aalto TK, Raivio KO.
Metabolism of extracellular adenine nucleotides by human endothelial cells exposed to reactive oxygen metabolites.
Am J Physiol.
1993;264:C282-C286 47. Picher M, Séviny J, D'Orléans-Juste P, Beaudoin AR. Hydrolysis of P2-purinoceptor agonists by a purified ectonucleotidase from the bovine aorta, the ATP-diphosphohydrolase. Biochem Pharmacol. 1996;51:1453-1460[Medline] [Order article via Infotrieve]. 48. Bo X, Fischer B, Maillard M, Jacobson KA, Burnstock G. Comparative studies on the affinities of ATP derivatives for P2X-purinoceptors in rat urinary bladder. Br J Pharmacol. 1994;112:1151-1159[Medline] [Order article via Infotrieve].
© 2000 by The American Society of Hematology.
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  X. Huang, D. J. Moore, R. J. Ketchum, C. S. Nunemaker, B. Kovatchev, A. L. McCall, and K. L. Brayman Resolving the Conundrum of Islet Transplantation by Linking Metabolic Dysregulation, Inflammation, and Immune Regulation Endocr. Rev., August 1, 2008; 29(5): 603 - 630. [Abstract] [Full Text] [PDF]
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F. Krotz, H. Y. Sohn, M. Keller, T. Gloe, S. S. Bolz, B. F. Becker, and U. Pohl Depolarization of Endothelial Cells Enhances Platelet Aggregation Through Oxidative Inactivation of Endothelial NTPDase Arterioscler Thromb Vasc Biol, December 1, 2002; 22(12): 2003 - 2009. [Abstract] [Full Text] [PDF]
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 K. Gounaris Nucleotidase Cascades Are Catalyzed by Secreted Proteins of the Parasitic Nematode Trichinella spiralis Infect. Immun., September 1, 2002; 70(9): 4917 - 4924. [Abstract] [Full Text] [PDF]
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N. C. Kaneider, P. Egger, S. Dunzendorfer, P. Noris, C. L. Balduini, D. Gritti, G. Ricevuti, and C. J. Wiedermann Reversal of Thrombin-Induced Deactivation of CD39/ATPDase in Endothelial Cells by HMG-CoA Reductase Inhibition: Effects on Rho-GTPase and Adenosine Nucleotide Metabolism Arterioscler Thromb Vasc Biol, June 1, 2002; 22(6): 894 - 900. [Abstract] [Full Text] [PDF]
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S. Ledoux, D. Laouari, M. Essig, I. Runembert, G. Trugnan, J.B. Michel, and G. Friedlander Lovastatin Enhances Ecto-5'-Nucleotidase Activity and Cell Surface Expression in Endothelial Cells: Implication of Rho-Family GTPases Circ. Res., March 8, 2002; 90(4): 420 - 427. [Abstract] [Full Text] [PDF]
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S. Ledoux, D. Laouari, M. Essig, I. Runembert, G. Trugnan, J.B. Michel, and G. Friedlander Lovastatin Enhances Ecto-5'-Nucleotidase Activity and Cell Surface Expression in Endothelial Cells: Implication of Rho-Family GTPases Circ. Res., March 8, 2002; 90(4): 420 - 427. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
-methylene] diphosphate (APCP), or by the addition of an A2a receptor
antagonist. Neither nitric oxide nor prostacyclin was involved in this
inhibitory activity, suggesting that Ado generated in the incubation
medium by the activity of 5'-NT on HUVECs inhibited platelet
aggregation. When ADP was incubated on HUVECs, it lost most of its
agonistic activity for platelets. Pretreatment of HUVECs with
APCP at a concentration that abolished ecto-5'-NT activity partially
restored ADP-induced platelet aggregation. Ecto-5'-NT contributes to EC function by inhibiting platelet aggregation in cooperation with ATP
diphosphohydrolase, which degrades ADP to AMP.
(Blood. 2000;96:2157-2162)
). Almost no Pi was liberated from ADP
preincubated on blank wells (open columns). The ADP-containing buffer
preincubated on HUVECs (
) lost its agonistic activity for platelets
in comparison with the ADP-containing buffer preincubated on blank
wells (
). The ADP-treated incubation buffer was added to PRP in a
10-fold dilution. The results are the mean ± SD of 3 separate
experiments.
), Pi was liberated from AMP on HUVECs in a dose-dependent manner
relative to the concentration of AMP added (
) and PGI2 (panel B, 
